Quantitative RT-PCR detection kit for ebola virus nucleic acid through one-step method

Abstract

The invention provides a quantitative RT-PCR detection kit for ebola virus nucleic acid through a one-step method. The kit comprises an RT-PCR reaction liquid, an RT-PCR enzyme mixture, an EBV (ebola virus) nucleic acid quantitative primer probe, an internal reference, negative control, critical positive control, strong positive control, and calibrators number 1 to number 5. According to the kit, the extracted EBV RNA can be subjected to one-step RT-PCR reaction; the EBV RNA in the sample can be subjected to fluorescence quantitative detection; the internal reference gene sequence is taken as the internal control; and contamination is prevented through UNG enzyme. The kit provided by the invention is simple in the one-step amplification method, simple and brief in program, simple and convenient to operate, capable of preventing contamination, high in specificity of the detection result, high in sensitivity, clear in result, high in credibility, and can be used for quantitative detection of EBV nucleic acid in serum.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to a biological detection technology, in particular to a kit for fluorescence quantitative detection of Ebola virus nucleic acid. Background technique [0002] Ebola virus (EBV) belongs to the Filoviridae family and is a severe infectious disease virus that can cause Ebola hemorrhagic fever (EBHF) in humans and primates. The incubation period of the virus can reach 2 to 21 days, but usually only 5 to 10 days; the disease can be fatal and includes several symptoms (including nausea, vomiting, diarrhea, changes in skin color, body aches, internal bleeding, external bleeding, Fever, etc.), the symptoms of infected people are very similar to those of Marburgvirus, which is also a member of the fibroviridae family, and has a 50% to 90% fatality rate. The main causes of death are stroke, myocardial infarction, hypovolemic shock or organ failure . EBHF was prevalent in the central African ...

AI Technical Summary

Problems solved by technology

At present, there are no EBV nucleic acid quantitative detection reagents on the market at home and abroad; the development of this kit can fill the gap in technology and products in this area

[0017] At the same time, in order to avoid false positives in the reaction results, using the anti-pollution characteristics of UNG enzyme, dUTP is used instead of dTTP in the PCR amplification reaction, so that only dUTP is contained in the amplified fragment; and dUTP makes the polluted amplicon amplified The target DNA is easily degraded by UNG enzyme: this dUTP-containing PCR product is incubated with UNG enzyme, because UDG enzyme can cleave the N-glycosyl bond between the uracil base and the sugar-phosphate backbone, and can be single-stranded or double-stranded Elimination of dUTP in DNA prevents Taq Elongation by DNA polymerase, thereby losing the ability to be reamplified